Multilayer hydrodynamic sheath flow structure

ABSTRACT

A microfabricated sheath flow structure for producing a sheath flow includes a primary sheath flow channel for conveying a sheath fluid, a sample inlet for injecting a sample into the sheath fluid in the primary sheath flow channel, a primary focusing region for focusing the sample within the sheath fluid and a secondary focusing region for providing additional focusing of the sample within the sheath fluid. The secondary focusing region may be formed by a flow channel intersecting the primary sheath flow channel to inject additional sheath fluid into the primary sheath flow channel from a selected direction. A sheath flow system may comprise a plurality of sheath flow structures operating in parallel on a microfluidic chip.

RELATED APPLICATIONS

The present invention is a continuation of U.S. patent application Ser.No. 16/719,537, filed on Dec. 18, 2019, which is a continuation of U.S.patent application Ser. No. 15/797,790, filed Oct. 30, 2017, now U.S.Pat. No. 10,543,992, which is a continuation of U.S. patent applicationSer. No. 15/269,556, filed Sep. 19, 2016 and now U.S. Pat. No.9,802,767, which is a continuation of U.S. patent application Ser. No.13/968,962, filed Aug. 16, 2013, now U.S. Pat. No. 9,446,912, which is acontinuation of U.S. patent application Ser. No. 13/179,084, filed Jul.8, 2011 and now U.S. Pat. No. 8,529,161, which is a continuation of U.S.patent application Ser. No. 12/610,753, filed Nov. 2, 2009 and now U.S.Pat. No. 7,997,831, which is a continuation application of U.S. patentapplication Ser. No. 11/998,557, filed Nov. 30, 2007 and now U.S. Pat.No. 7,611,309, which, in turn, is a continuation application of U.S.patent application Ser. No. 10/979,848 entitled “Multilayer HydrodynamicSheath Flow Structure,” filed Nov. 1, 2004 and now U.S. Pat. No.7,311,476, which claims priority to U.S. Provisional Application Ser.No. 60/516,033, filed Oct. 30, 2003. The entire contents of each of theabove applications are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to a system and method for producing asheath flow in a flow channel. More particularly, the present inventionrelates to a system and method for producing a sheath flow in amicrochannel in a microfluidic device.

BACKGROUND OF THE INVENTION

Sheath flow is a particular type of laminar flow in which one layer offluid, or a particle, is surrounded by another layer of fluid on morethan one side. The process of confining a particle stream in a fluid isreferred to as a ‘sheath flow’ configuration. For example, in sheathflow, a sheath fluid may envelop and pinch a sample fluid containing anumber of particles. The flow of the sheath fluid containing particlessuspended therein may be narrowed almost to the outer diameter ofparticles in the center of the sheath fluid. The resulting sheath flowflows in a laminar state within an orifice or channel so that theparticles are lined and accurately pass through the orifice or channelin a single file row.

Sheath flow is used in many applications where it is preferable toprotect particles or fluids by a layer of sheath fluid, for example inapplications wherein it is necessary to protect particles from air. Forexample, particle sorting systems, flow cytometers and other systems foranalyzing a sample, particles to be sorted or analyzed are usuallysupplied to a measurement position in a central fluid current, which issurrounded by a particle free liquid sheath.

Sheath flow is useful because it can position particles with respect tosensors or other components and prevent particles in the center fluid,which is surrounded by the sheath fluid, from touching the sides of theflow channel and thereby prevents clogging of the channel Sheath flowallows for faster flow velocities and higher throughput of samplematerial. Faster flow velocity is possible without shredding cells inthe center fluid because the sheath fluid protects the cells from shearforces at the walls of the flow channel.

Conventional devices that have been employed to implement sheath flowhave relatively complex designs and are relatively difficult tofabricate.

SUMMARY OF THE INVENTION

The present invention provides a microfabricated sheath flow structurefor producing a sheath flow for a particle sorting system or othermicrofluidic system. The sheath flow structure may comprise a two-layerconstruction including a sheath inlet for introducing a sheath fluidinto a primary sheath flow channel and a sample inlet for introducing asample to the structure. A sample is introduced to the sheath fluid inthe primary sheath flow channel via the sample inlet and suspendedtherein. The primary sheath flow channel may branch at a locationupstream of a sample inlet to create a flow in an upper sheath channel.The primary sheath flow channel forms a primary focusing region foraccelerating sheath fluid in the vicinity of a sample channel connectedto the sample inlet. The sample channel provides the injected sample tothe accelerating region, such that the particles are confined in thesheath fluid. The primary focusing region further focuses the sheathfluid around the sample. The sheath flow then flows to a secondarysheath region downstream of the primary accelerating region connects theupper sheath channel to the primary sheath flow channel to further focusthe sample in the sheath fluid. The resulting sheath flow forms afocused core of sample within a channel.

The sheath flow structure may be parallelized to provide a plurality ofsheath flow structures operating in parallel in a single system. Theparallelized system may have a single sample inlet that branches into aplurality of sample channels to inject sample into each primary sheathflow channel of the system. The sample inlet may be provided upstream ofthe sheath inlet. Alternatively, the parallelized system may havemultiple sample inlets. The parallelized sheath flow structure may havea single sheath fluid inlet for providing sheath fluid to all of theprimary sheath flow channels and/or secondary sheath channels, ormultiple sheath fluid inlets for separately providing sheath fluid tothe primary sheath flow channels and or secondary sheath channels.

According to a first aspect of the invention, a sheath flow structurefor suspending a particle in a sheath fluid is provided. The sheath flowstructure comprises a primary sheath flow channel for conveying a sheathfluid, a sample inlet for injecting a particle into the sheath fluidconveyed through the primary sheath flow channel, a primary focusingregion for focusing the sheath fluid around the particle in at least afirst direction and a secondary focusing region provided downstream ofthe primary focusing region. The secondary focusing region focuses thesheath fluid around the particle in at least a second directiondifferent from the first direction.

According to another aspect of the invention, a sheath flow structurefor suspending a particle in a sheath fluid comprises a first substratelayer including a primary sheath flow channel for conveying a sheathfluid and a second substrate layer stacked on the first substrate layer.The second substrate layer includes a first sheath inlet for introducinga sheath fluid to the primary sheath flow channel, a sample inletdownstream of the first sheath inlet for providing the particle to theprimary sheath flow channel in a primary focusing region to form asheath flow including the particle surrounded by the sheath fluid on atleast one side. A first secondary sheath channel is formed in the firstor second substrate layer in communication with the primary sheath flowchannel. The first secondary sheath channel diverts a portion of saidsheath fluid from the primary sheath flow channel.

According to still another aspect of the invention, a focusing regionfor focusing a particle suspended in a sheath fluid in a channel of asheath flow device is provided. The focusing region comprises a primaryflow channel for conveying a particle suspended in a sheath fluid and afirst secondary flow channel intersecting the primary flow path forinjecting sheath fluid into the primary flow channel from above theparticle to focus the particle away from a top wall of the primary flowchannel.

According to another aspect of the invention, a method of surrounding aparticle on at least two sides by a sheath fluid, comprises the steps ofinjecting a sheath fluid into a primary sheath flow channel diverting aportion of the sheath fluid into a branching sheath channel, injectingthe particle into the primary sheath flow channel to suspend theparticle in the sheath fluid to form a sheath flow and injecting thediverted portion of the sheath fluid into the sheath flow to focus theparticle within the sheath fluid.

According to another aspect of the invention, a method of surrounding aparticle on at least two sides by a sheath fluid, comprises the steps ofconveying a sheath fluid through a primary sheath flow channel,injecting a particle into the sheath fluid conveyed through the primarysheath flow channel, focusing the sheath fluid around the particle in atleast a first direction and focusing the sheath fluid around theparticle in at least a second direction different from the firstdirection.

According to still another aspect, a sheath flow system is providedwhich comprises a plurality of a sheath flow structures operating inparallel on a substrate. Each sheath flow structure comprises a primarysheath flow channel for conveying a sheath fluid, a sample channel forinjecting a particle into the sheath fluid conveyed through the primarysheath flow channel, a primary focusing region for focusing the sheathfluid around the particle in at least a first direction and a secondaryfocusing region provided downstream of the primary focusing region forfocusing the sheath fluid around the particle in at least a seconddirection different from the first direction.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a sheath flow structure according to an illustrativeembodiment of the invention.

FIGS. 2A-2B illustrate a multilayer sheath flow structure according toan illustrative embodiment of the invention.

FIG. 2C illustrates is a cross sectional view through the centerline ofthe sheath flow structure of FIG. 2A, showing the path of an injectedparticle through the structure.

FIG. 3 illustrates the path of a particle through the multilayer sheathflow structure of FIGS. 2A-2C.

FIG. 4A illustrates the flow profile within the primary focusing regionand the secondary focusing region during operation of the sheath flowstructure of FIGS. 2A-2C.

FIGS. 4B-4D are detailed cross-sectional views of the flow profileswithin the primary sheath flow channel at different stages duringoperation of the sheath flow structure of FIGS. 2A-2C.

FIGS. 5A-5C illustrates a multilayer sheath flow structure according toan alternate embodiment of the invention, where a sample is injecteddirectly into a focusing region.

FIG. 6 is a perspective view of a sheath flow structure according toanother embodiment of the invention.

FIGS. 7A-7B illustrate a sheath flow structure including a sample inletprovided upstream of a sheath flow inlet according to another embodimentof the invention.

FIGS. 8A-8B illustrate a parallelized sheath flow system for producingsheath flow in multiple parallel channels according to anotherembodiment of the invention.

FIG. 9A is a fluorescent microscope image of a primary sheath flowchannel downstream from the secondary focusing region in theparallelized sheath flow system of FIGS. 8A and 8B.

FIG. 9B is fluorescent microscope image taken of a sample in the primarysheath flow channel of FIG. 9A after focusing of the sample.

FIG. 10 is a histogram diagramming the measured amount of fluorescencein the channel observed in FIG. 9B across axis -A-A-.

FIG. 11 is a histogram superimposing the fluorescence measurements fromall eight primary sheath flow channels in the system of FIGS. 8A-8B.

FIG. 12 illustrates the distribution of core sizes for the sheath flowsproduced in the primary sheath flow channels in the system of FIGS.8A-8B.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a system and method for producing asheath flow in a flow channel, such as a microchannel. The presentinvention will be described below relative to illustrative embodiments.Those skilled in the art will appreciate that the present invention maybe implemented in a number of different applications and embodiments andis not specifically limited in its application to the particularembodiments depicted herein.

As used herein, the term “microfluidic” refers to a system or device forhandling, processing, ejecting and/or analyzing a fluid sample includingat least one channel having microscale dimensions.

The terms “channel” and “flow channel” as used herein refers to apathway formed in or through a medium that allows for movement offluids, such as liquids and gases. A “microchannel” refers to a channelin the microfluidic system preferably have cross-sectional dimensions inthe range between about 1.0 μm and about 500 μn, preferably betweenabout 25 μn and about 250 μn and most preferably between about 50 μm andabout 150 μn. One of ordinary skill in the art will be able to determinean appropriate volume and length of the flow channel. The ranges areintended to include the above-recited values as upper or lower limits.The flow channel can have any selected shape or arrangement, examples ofwhich include a linear or non-linear configuration and a U-shapedconfiguration.

FIG. 1 illustrates a microfabricated sheath flow structure 10 accordingto an illustrative embodiment of the invention. The sheath flowstructure 10 may be used to suspend particles in a sheath fluid flowstream for use in a particle sorting system or other microfluidicsystem. The sheath flow structure 10 includes a primary sheath flowchannel 12 for conveying sheath fluid through the sheath flow structure10. A flow may be induced through the primary sheath flow channel 12through any means known in the art, including one or more pumps. Thesheath flow structure 10 further includes a sample inlet 15 forintroducing a sample, such as one or more particles, to the sheath fluidflowing through the primary sheath flow channel 12, so that the sampleis surrounded by the flowing sheath fluid. The sample inlet 15 maycomprise a channel, reservoir or other suitable component incommunication with the primary sheath flow channel 12.

According to one embodiment, the microfabricated sheath flow structureis formed on a microfluidic chip and the primary sheath flow channel andother flow channels formed therein are microchannels having microscaledimensions. However, one skilled in the art will recognize that thesheath flow structure may alternatively have larger dimensions and beformed using flow channels having cross-sectional dimensions greaterthan 500 μm. The illustrative sheath flow structure can be fabricated inglass, plastics, metals or any other suitable material usingmicrofabrication, injection molding/stamping, machining or othersuitable fabrication technique.

After introduction of the sample into the sheath fluid, a primaryfocusing region 17 accelerates and focuses the sheath fluid around theinjected sample. Preferably, the primary focusing region 17 focuses thesheath fluid away from the sides and bottom of the sample. A secondaryfocusing region 19, disposed downstream of the primary focusing region17 along the primary sheath flow channel, provides additional focusingof the sheath fluid around the sample after the primary focusing regionperforms the primary focusing. Preferably, the secondary focusing region19 focuses the sample in a vertical direction from above the sample.

According to an illustrative embodiment, the combination of the primaryfocusing region 17 and the secondary focusing region 19 providesthree-dimensional focusing of the sheath fluid around the sample. Theresulting sheath flow is sample-focused hydrodynamically on all sides ofthe sample away from the walls of the primary sheath flow channel 12,with the sample being suspended as a focused core in the approximatecenter of the channel.

The secondary focusing region 19 passes the resulting sheath flow in theprimary sheath flow channel 12 to a particle sorting system or othermicrofluidic system or component in fluid communication with an outlet19 a of the secondary focusing region 19. The microfluidic system forreceiving the sheath flow may be formed on the same chip or substrate asthe sheath flow structure or a different substrate in fluidcommunication with the sheath flow structure 10.

According to one embodiment, the sheath flow structure may be formedusing a plurality of stacked layers. For example, FIGS. 2A-2C illustratea two-layer sheath flow structure 100 for producing sheath flowaccording to one embodiment of the invention. In FIGS. 1 and 2A-2C,similar parts are indicated by equivalent reference numbers. Theillustrated sheath flow structure 100 has a two-layer constructionincluding a bottom substrate layer 10 b and a top substrate layer 10 astacked on the bottom substrate layer 10 b. Those of ordinary skill willrecognize that any suitable number of layers can be used. The topsubstrate layer 10 a may have formed therein a sheath inlet 11 forintroducing a sheath fluid to the primary sheath flow channel 12 and asample inlet 15 for introducing a sample to the sheath flow structure.The primary sheath flow channel 12 for conveying the sheath fluidthrough the structure is formed in the bottom layer 10 b of thetwo-layer sheath flow structure 100. As shown, the sample inlet 15connects to a sample channel 16, which intersects the primary sheathflow channel 12 downstream of the sheath inlet 11 to inject a sample,such as a stream of particles, into a sheath fluid flowing in theprimary sheath flow channel 12.

While the illustrative two-layer sheath flow structure 100 injects thesheath flow and sample particles from a top surface of the structure,one skilled in the art will recognize that the sheath inlet 11 andsample inlet 15 can be provided in any suitable location and have anysuitable size and configuration.

The primary focusing region 17 in the two-layer sheath flow structure100 of FIGS. 2A-2C may be formed by tapering the primary sheath flowchannel 12 from a relatively wide width W to a smaller width W′downstream of the intersection between the sample channel 16 and theprimary sheath flow channel 12, as shown in FIG. 2B. The height of thechannel may be substantially constant throughout the length of thechannel or may be varied to facilitate focusing of the sample within thesheath fluid.

In the embodiment shown in FIG. 2A, the primary focusing region 17 isformed by dividing the primary sheath flow channel 12 into twosubchannels 12 a, 12 b upstream from the sample inlet 15. The divergingsubchannels 12 a, 12 b form a sample injection island 50 therebetween.At the downstream end of the sample injection island 50, the subchannels12 a, 12 b merge to form the primary focusing region 17. The sample flowchannel 16 projects into the primary focusing region 17 to convey thesample particles provided via the sample inlet 15 to the primaryfocusing region 17, such that the sample particles are suspended in thesheath fluid. Alternatively, each of the subchannels 12 a, 12 b may havea separate inlet, and the separated subchannels may converge in theprimary focusing region 17.

In the primary focusing region 17, the sample particles injected intothe sheath flow are focused away from the sides and bottom by the sheathflow. As shown, the outlet of the sample flow channel 16 is insubstantially the middle of the primary focusing region 17, between theoutlets of the subchannels 12 a, 12 b, such that the particles aresurrounded by sheath fluid flowing from the subchannels on both sides ofthe injected particles and centralized within the sheath fluid flow. Thesheath flow channel 12 in the primary focusing region then tapers from arelatively wide width W at the outlets of the subchannels 12 a, 12 b toa smaller width W′ to force the sheath fluid around the suspended sampleparticles.

After suspension of the sample particles, the sheath flow then flowsfrom the primary focusing region 17 through the sheath flow channel 12,which forms the secondary focusing region 19 downstream of the primaryfocusing region 17. According to an illustrative embodiment, thesecondary focusing region 19 utilizes sheath fluid to provide secondaryfocusing of the sheath flow in a vertical direction after the initialfocusing provided by the primary focusing region 17. For example, asshown in FIGS. 2A-2C, the secondary focusing region 19 may be formed bysecondary sheath channels 13 a, 13 b that intersect the primary sheathflow channel 12 in the secondary focusing region 19. The secondarysheath channels 13 a, 13 b flow and inject sheath fluid into the primarysheath flow channel 12 to focus the sample within the sheath fluid.

As shown, the inlets to the secondary sheath channels 13 a, 13 b,respectively, may intersect the primary sheath flow channel 12 in anintermediate upstream region between the sheath inlet 11 and the outletof the sample channel 16. Branch points 24 a, 24 b connect each of thesecondary sheath channels 13 a, 13 b to the primary channel 12 to diverta portion of the sheath fluid from the primary sheath flow channel toeach of the secondary sheath channels 13 a, 13 b, respectively. Thediverted sheath flow then flows to the secondary focusing region 19,where the outlets of the secondary sheath channels 13 a, 13 b intersectthe primary sheath flow channel 12. Preferably, the outlets of bothsecondary sheath channels extend above and substantially parallel to thefluid flow in the primary sheath flow channel 12 in the vicinity of thesecondary focusing region 19. In this manner, secondary sheath fluidfrom the secondary sheath channels 13 a, 13 b enters the primary sheathflow channel 12 from the same side as the sample, compressing thesuspended sample away from the upper wall of the channel 12 (i.e., inthe other direction from the main sheath of fluid around the particle).

In the illustrative embodiment, branch points 24 a, 24 b extendsubstantially transverse or perpendicular to the primary sheath flowchannel, while sheath channels 13 a, 13 b connected to the branch points24 a, 24 b, respectively, extend substantially parallel to the primarysheath flow channel 12. Connection branches 25 a, 25 b for connectingthe sheath channels 13 a, 13 b, respectively, to the primary sheath flowchannel in the secondary focusing region 19 may be parallel to thebranch points 24 a, 24 b to create a flow path that is substantiallyreverse to the direction of the flow path through the branch points 24a, 24 b, while the outlets inject the secondary sheath fluid along apath that is above and substantially parallel to fluid flow in theprimary sheath flow channel 12.

In the embodiment of FIGS. 2A-2C, the secondary sheath channels 13 a, 13b are formed in the upper substrate layer 10 a and placed intocommunication with the primary sheath flow channel 12 in the lowersubstrate layer 10 b when the upper substrate layer 10 a is stacked onthe lower substrate layer. However, in another embodiment of theinvention, one or both of the secondary sheath channels can be formed inthe lower substrate layer to provide focusing from any suitabledirection.

While the illustrative embodiment includes two branch points 24 a, 24 b,each connecting to a respective secondary sheath flow channel 13 a, 13 bextending on opposite sides of the primary sheath flow channel 12, oneskilled in the art will recognize that the sheath flow structure of thepresent invention may include any suitable number of secondary sheathchannels having any suitable size, location and configuration.

FIG. 2C is a cross-sectional side view of the sheath flow structure 100comprising a stacked upper substrate layer and a lower substrate layer.As shown, the primary sheath flow channel may be formed as an openchannel in a top surface of the lower substrate layer 10 b. The sheathinlet 11 and the sample inlet 15 each extend through the upper substratelayer 10 a from one surface 102 of the upper substrate layer 10 a to theopposite surface 103. When the upper substrate layer 10 a is stacked onthe lower substrate layer 10 b, the sheath inlet 11 and sample inlet 15are placed in communication with the primary sheath flow channel 12. Thebottom surface 103 of the upper substrate layer 10 a may further serveto enclose the primary sheath flow channel 12 when the two substratelayers are stacked together. As also shown, the stacking of the uppersubstrate layer places the inlet and outlet of each of the secondarysheath channels 13 a, 13 b in communication with the primary sheath flowchannel 12.

The substrate layers 10 a, 10 b can be machined, molded or etched toform the channels inlets and focusing regions. Suitable materials forforming the substrates 10 a, 10 b include, but are not limited tosilicon wafer, plastic, glass and other materials known in the art.

FIG. 3 is a cross-sectional view of the sheath flow structure 100illustrating the path of a sample particle injected into the sheath flowstructure according to the teachings of the present invention. FIG. 4Ais a perspective cross-sectional view of the sheath flow structure 100illustrating the sheath fluid and suspending particle during thedifferent stages of producing a sheath flow. FIGS. 4B-4D arecross-sectional detailed views of the primary flow channel 12 during thedifferent stages of producing a sheath flow. As shown in FIG. 4B, sample160 from the sample inlet 15 enters the primary focusing region 17through the sample channel 16 connected to the sample inlet 15 and isfocused on three sides by accelerating sheath fluid 120 flowing from thesecondary sheath channels into the sheath channel 12 in the primaryfocusing region 17. The resulting focused flow 170, having the particlessuspended therein, passes to the secondary focusing region 19.Additional sheath fluid 130 enters the primary sheath flow channel 12through a connector in the secondary focusing region 19 to focus thesuspended particles on the fourth side, forming a central core 190, asshown in detail in FIG. 4C. The resulting sheath flow 200 is a laminarflow that is sample focused hydrodynamically from all sides away fromthe walls at the channel center, as shown in FIG. 4D. The desirable coreflow location may or may not be at center of the primary sheath flowchannel downstream of the secondary sheath flow structure.

In the illustrative embodiment, the flow resistance ratio between theprimary sheath flow channel 12 and the branched secondary sheathchannels 13 a, 13 b is calibrated to position the core at specificregion in the downstream sheath flow channel. The desirable core flowlocation may or may not be at center of downstream channel.

According to an alternate embodiment of the invention, shown in FIGS.5A-5C, in which like parts are indicated by like reference numbers, thesample can be injected directly from a sample inlet 15 b into a primaryfocusing region 17′, which is formed by a widening of the sheath channel12 downstream of the branch points 24 a, 24 b. The primary sheath flowchannel 12 conveys the sheath fluid directly to the primary focusingregion 17, and the sample particles are directly injected into thecenter of the sample flow and confined therein. In this embodiment, theprimary sheath flow channel does not branch into subchannels, and theacceleration of the sheath fluid and suspension of injected particlescan be accomplished by shaping the primary sheath flow channel in asuitable manner.

FIG. 6 illustrates a sheath flow structure 100 according to anotherembodiment of the invention, including separate sheath inlets for theprimary sheath flow and the sheath fluid (secondary sheath fluid) thatis added to the sheath flow in the secondary focusing region 19. Asshown, the sheath flow structure 100 of FIG. 5 includes a primary sheathinlet 11 a for providing a primary sheath flow to suspend the injectedsample particles and a secondary sheath inlet 11 c for providingsecondary sheath flow to focus the particles within the primary sheathfluid in the secondary focusing region. In the embodiment shown in FIG.5 , the primary sheath inlet 11 c is formed in an upper substrate layer10 a and the secondary sheath inlet 11 c is formed in a second substratelayer 10 b, though one skilled in the art will recognize that theinvention is not limited to this configuration.

According to another embodiment of the invention, shown in FIGS. 7A and7B, the sample inlet 15 may be provided upstream or behind the sheathinlet. In this embodiment, the upstream portion of the primary sheathflow channel 12 comprises two separate subchannels 12 a, 12 b, whichconverge in the primary focusing region 17. Each subchannel 12 a, 12 bhas a separate inlet 11 a, 11 b for introducing sheath fluid to therespective subchannel. The embodiment of FIGS. 7A and 7B furtherincludes separate sheath inlets 11 c, 11 d for the secondary sheathchannels 13 a, 13 b. As described above, the secondary sheath channelsintersect the primary sheath flow channel 12 in the secondary focusingregion to provide focusing of a sample within a flowing sheath fluid inthe primary sheath flow channel 12. The design of the illustrativesheath flow structure of FIGS. 7A and 7B is suitable for parallelizationof the sheath flow process because multiple sample channels 16 can befed into an array of sheath fluid injectors on a single microfluidicchip.

While the embodiment of FIG. 7 shows separate sheath inlets for eachsubchannel of the primary flow channel and each secondary sheath flowchannel, one skilled in the art will recognize that the primary sheathflow channel can alternatively have a single inlet, as shown in FIGS.2A-2C, 5A-5C and 6 . The primary sheath flow channel can includesubchannels that converge to suspend an injected particle, as describedwith respect to FIGS. 2A-2C and 6 . The primary sheath flow channel mayalternatively be shaped and configured to widen to surround an injectedparticle, as described with respect to FIGS. 5A-5C. In addition, whilethe embodiment of FIG. 7 shows the secondary sheath flow channels to beformed separately from the primary sheath flow channel, one skilled inthe art will also recognize that one or more of the secondary sheathflow channels 13 a, 13 b may be formed by diverted a portion of thesheath fluid in the primary sheath flow channel into one or more of thesecondary flow channels, eliminating the need for a separate sheathinlet.

FIGS. 8A-8B illustrate an array of sheath flow structures 10 a-10 h maybe formed on a single microfluidic chip 800 according to anotherembodiment of the invention. The microfluidic chip 800 can comprise anupper substrate layer including selected components of each sheath flowstructure and a lower substrate layer including selected components ofeach sheath flow structures such that when the upper substrate layer isstacked on the lower substrate layer, the array of sheath flowstructures is formed. FIG. 8 illustrates an array of eight parallelthree-dimensional sheath flow structures 10 a-10 h implementing the rearsample injection scheme of FIG. 7 . As shown, a single sample inlet 15can be used to inject a sample into each of the primary sheath flowchannels 12 a-12 h. The microfabricated design allows the system toprecisely split an input sample provided in the sample inlet among eightseparate sample channels 16 a-16 h, which then inject the sample intothe primary sheath flow channels. The use of a sample inlet 15 upstreamof the sheath inlet facilitates parallelization of multiple sheath flowstructures in a single integrated system. Alternatively, a sample inletprovided upstream of the sheath flow inlets may be separately providedfor each primary sheath flow channel.

Each of the channel inlets 11 a, 11 b, 11 c or 11 d for each sheath flowstructure may be aligned, as shown in FIGS. 8A and 8B or staggered.Furthermore, a single inlet may be provided for one or more primarysheath flow channel and/or secondary sheath channel in each sheath flowstructure in the parallelized system of FIG. 8 , or the channels mayshare inlets, as described above.

In the embodiment shown in FIG. 8B, the primary sheath flow channels 12a-12 h converge downstream of the secondary focusing regions, so thatthe sheath flows produced therein are rejoined and flowed off-chip via asingle outlet 812. Alternatively, each primary sheath flow channel canseparately flow off-chip.

EXEMPLIFICATION OF THE INVENTION

The parallelized sheath flow structure 800 of FIG. 8 was formed on amicrofluidic chip and used to produce a sheath flow. Eight primarysheath flow channels were formed 800 microns apart, with associatedsample channels, secondary sheath flow channels and other componentsalso formed in parallel on the chip. The chip was glued to a fixture by300LSE adhesive available from 3M Corporation and cured for 72 hours. A10:1 dilution of 6 micron yellow beads from Spherotech was used as thesample and DakoCytomation sheath buffer was used as sheath fluid. Thesheath fluid to sample ratio was 45:1. The flow rate was produced sothat the number of beads flowing through a selected primary sheath flowchannel was about 750 beads per second. The injected sample was dividedamong the eight sample channels and the sample portion in each samplechannel was injected into the DakoCytomation sheath buffer flowingthrough the primary sheath flow channel associated with that samplechannel. The sheath flow was initially focused from the sides and bottomof the channel in the primary focusing regions around the sample of eachprimary sheath flow channel. After the primary focusing, the sampleflowed to the secondary focusing region where sheath fluid from thesecondary sheath channels was injected to focus the sample in a verticaldirection and form a core of the sample within each primary sheath flowchannel.

The resulting sheath flow was then observed using a fluorescentmicroscope over a period of about eight seconds, and the results areshown in FIGS. 9A-10 . FIG. 9A is an image of one of the primary sheathflow channels 12 in the system of FIG. 8 , illustrating the side walls111, 112 of the channel 12. FIG. 9B is a fluorescent microscope image ofthe same region of the channel as shown in FIG. 9A taken using thefluorescent microscope after the secondary focusing of the sample in thechannel. The side walls, while not clearly visible in FIG. 9B are in theapproximate same location within the Figure as are the side walls 111,112 in FIG. 9A. The bright spot illustrates the concentration of thefluorescent beads 160 of the sample in the core ten microns of thetwo-hundred micron channel 12. FIG. 10 is a histogram of the image ofFIG. 9B across axis -A-A- demonstrating the clean core flow produced bythe sheath flow structure. The magnitude of the peak in the histogramsreflects the amount of fluorescence observed for each location withinthe respective channel. As clearly shown in these Figures, the sheathflow structure 800 of the illustrative embodiment of the inventionproduces a sample-focused hydrodynamic sheath flow forming a central,focused core of sample 160 within a channel 12.

FIG. 11 is a histogram comparing the cores for each of the resultingsheath flows in all eight samples of in the parallelized sheath flowstructure of FIG. 8 . As shown, each channel produces a substantiallysimilar central core of the sample within the sheath fluid. The core isproduced in substantially the same location within each primary sheathflow channel. The approximate locations of the side walls of thechannels are indicated by reference numbers 111, 112.

FIG. 12 illustrates the distribution of core sizes from the single eightprimary sheath flow channels in the test system of FIG. 8 . As shown,the core produced in all of the channels using the sheath flowproduction method described above falls within 8.8+/−0.7 micron corewidth.

The sheath flow structure of the illustrative embodiment of theinvention provides significant advantages not found in sheath flowstructures of the prior art. The illustrative sheath flow structureprovides three-dimensional hydrodynamic focusing using a single sheathfluid inlet. The illustrative sheath flow structure has a compactstructure designed for manufacturability and requires only twostructural layers in fabrication. Because the entrance to the sheathflow channels are only required on one side of the structure, thefluidic input/output structures can be simplified. Furthermore, the coreflow vertical location is controllable by geometric (lithographic)resistance ratios between adjacent channels. The illustrative sheathflow structure provides accurate results that are largely insensitive toalignment between adjacent layers, as the only alignment required is tomaintain the components in adjacent layers along the same centerline.The reentrant flow downstream of sample injection is then symmetric. Inaddition, the long path length of the branching upper sheath channels 13a, 13 b results in negligible resistance ratio (therefore flow rateratio) shift between two branch arms through misalignment ofcenterlines.

The present invention has been described relative to an illustrativeembodiment. Since certain changes may be made in the above constructionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description or shown in theaccompanying drawings be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are to cover allgeneric and specific features of the invention described herein, and allstatements of the scope of the invention which, as a matter of language,might be said to fall therebetween.

The invention claimed is:
 1. A method of sorting cells, comprising thesteps of: conveying a sheath fluid through a primary sheath flowchannel; obtaining a sample of cells; injecting the sample into thesheath fluid using a sample inlet intersecting the primary sheath flowchannel; focusing the sample in at least a vertical direction in aprimary focusing region downstream of the sample inlet; focusing thesample in at least a horizontal direction in a secondary focusing regiondownstream of the primary focusing region; sorting the cells in thesample downstream of the secondary focusing region; and collecting thesorted cells.
 2. The method of claim 1, further comprising focusing thesample in a horizontal direction in the primary focusing region.
 3. Themethod of claim 1, further comprising focusing the sample in a verticaldirection in the secondary focusing region.
 4. The method of claim 1,wherein a height of the primary sheath flow channel is reduced in theprimary focusing region to produce focusing of the sample in at leastthe vertical direction.
 5. The method of claim 3, wherein a height ofthe primary sheath flow channel is reduced in the secondary focusingregion to produce focusing of the sample in the vertical direction. 6.The method of claim 5, wherein the reduction in height in the secondaryfocusing region is caused by a taper in the primary sheath flow channel.7. The method of claim 1, wherein the primary sheath flow channel tapersin the secondary focusing region to produce the focusing of the samplein the horizontal direction.
 8. The method of claim 2, wherein theprimary sheath flow channel tapers in the primary focusing region toproduce the focusing of the sample in the horizontal direction.
 9. Themethod of claim 1, wherein sorting the cells further comprises using aparticle sorting system operatively coupled to the primary sheath flowchannel.
 10. The method of claim 1, further comprising analyzing thecells downstream of the secondary focusing region using a detectoroperatively coupled to the primary sheath flow channel.
 11. The methodof claim 1, wherein a height and a width of the primary sheath flowchannel reduce to between 50 and 150 micrometers at an end of thesecondary focusing region thereby to focus the stream of cellsvertically and horizontally.
 12. The method of claim 1, wherein a heightof the primary sheath flow channel is reduced in the primary focusingregion by changing a height of a top channel wall and a height of abottom channel wall.
 13. The method of claim 1, wherein a height of theprimary sheath flow channel is reduced in the secondary focusing regionby changing a height of a top channel wall and a height of a bottomchannel wall.